Expression, purification and characterization of recombinant human beta-amyloid 1-42 in Pichia pastoris.
The human peptide rhA beta(1-42) was effectively produced through a novel expression system and purification procedure. The peptide rhA beta(1-42) was successfully expressed in Pichia pastoris, the methylotrophic yeast that has never been used as host. The cDNA encoding full-length hA beta(1-42) was synthesized with yeast bias codons and cloned into the pPICZ alpha A vector in frame with the yeast alpha-factor secretion signal under the transcriptional control of the AOX1 promoter and integrated into the secreting expression organism P. pastoris strain X33. Production of rhA beta(1-42) through fermentation was further optimized and scaled up in an 80 L fermentor. Secreted rhA beta(1-42) was purified using a two-step purification scheme: SP Sepharose ion exchange chromatography and source 30 RPC. The purification procedure is fast and efficient and reached a recovery of >93% without loss of activity. The purified rhA beta(1-42) was confirmed by Western blotting analysis and N-terminals amino sequencing analysis. This efficient and cost-effective expression system facilitates large-scale production and purification for recombinant rhA beta(1-42).
Shen
Mohan
M
Department of Biological Engineering, College of Pharmacy & Institute of Frontier Medical Sciences, Jilin University, 1163 Xin Min Street, Changchun, Jilin 130021, PR China.
Wang
Quancai
Q
Mu
Xupeng
X
Xu
Hao
H
Yan
Weiqun
W
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000302:isolation & purification*; D001483:Base Sequence; D019149:Bioreactors; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C075222:amyloid beta-protein (1-42)